Core A provides unique expertise with multiphoton microscopy for in vivo imaging, cell collection and analysis and unique expertise with the application of fluorescence in situ hybridization (FISH) and microarray analysis at the single cell level (CHIP). We have developed imaging methods to adapt the multi-photon microscope for use with either rats or mice. The method requires the stable expression of either GFP, CFP or YFP in carcinoma cells, macrophages and endothelial cells within the primary tumor. This method can be used to image cells within primary tumors resulting from cell grafts and transgenic mice bearing primary tumors that develop as the result of mammary epithelial cell specific expression of oncogenes. We have optimized the surgical removal of the overlying skin to allow more direct contact between the objective and tumor surface in the live animal without disrupting the integrity of the primary tumor, requiring a viewing window or producing a wounding response as monitored by imaging of GFP-expressing white cells. Multiphoton microscope-based imaging of whole animals will be performed to record cell behavior including cell motility, adhesion and intravasatation in intact living non-metastatic and metastatic tumors in animals with different genetic backgrounds. Multiphoton second harmonic excitation will be used to image extracellular matrix in the living tumor so as to identify the sites of proteolysis and cell adhesion. The isolation of subpopulations of live invasive cells from the primary tumor using chemotaxis of the invasive carcinoma cells into microneedles is also part of this core. This unique method will allow the collection, study in primary culture and gene expression analysis of the invasive subpopulation of cells in the primary tumor. FISH & CHIPs technology fuses functional genomics with single cell analysis. The approach uses information from the genome sequence to generate synthetic DNA probes conjugated to various fluorochromes, and thereby to create a "barcode" that allows identification of multiple species of RNAs within a single cell "a transcriptome". The fluorescent probes are used to hybridize to the RNAs in situ (hence FISH: fluorescence in situ hybridization). The site where each RNA species is most concentrated is where it is being synthesized: the transcription site. This is the most direct evidence that a specific gene is active. By using multiple barcodes, many genes (e.g. 11) can be characterized as off or on by detecting this transcription site in each cell, This technology will allow identification of which cells are activated by EGF and CSF-1 and the expression patterns of individual cells in situ. This core will support the use of these methods by all of the projects of this PPG.